In the fields of molecular biology and biochemistry, as well as in the diagnosis of diseases, nucleic acid hybridization has become a powerful tool for the detection, isolation and analysis of specific oligonucleotide sequences. Typically, such hybridization assays utilize an oligodeoxynucleotide probe that has been immobilized on a solid support; as for example in the reverse dot blot procedure (Saiki, R. K., Walsh, P. S., Levenson, C. H., and Erlich, H. A. (1989) Proc. Natl. Acad. Sci. USA 86, 6230). More recently, arrays of immobilized DNA probes attached to a solid surface have been developed for sequencing by hybridization (SBH) (Drmanac, R., Labat, I., Brukner, I., and Crkvenjakov, R. (1989) Genomics, 4, 114-128), (Strezoska, Z., Pauneska, T., Radosavljevic, D., Labat, I., Drmanac, R., and Crkvenjakov, R. (1991) Proc. Natl. Acad. Sci. USA, 88, 10089-10093). SBH uses an ordered array of immobilized oligodeoxynucleotides on a solid support. A sample of unknown DNA is applied to the array, and the hybridization pattern is observed and analyzed to produce many short bits of sequence information simultaneously. An enhanced version of SBH, termed positional SBH (PSBH), has been developed which uses duplex probes containing single-stranded 3xe2x80x2-overhangs. (Broude, N. E., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Proc. Natl. Acad. Sci. USA, 91, 3072-3076). It is now possible to combine a PSBH capture approach with conventional Sanger sequencing to produce sequencing ladders detectable, for example by gel electrophoresis (Fu, D., Broude, N. E., Kxc3x6ster, H., Smith, C. L. and Cantor, C. R. (1995) Proc. Natl. Acad. Sci. USA 92, 10162-10166).
For the arrays utilized in these schemes, there are a number of criteria which must be met for successful performance. For example, the immobilized DNA must be stable and not desorb during hybridization, washing or analysis. The density of the immobilized oligodeoxynucleotide must be sufficient for the ensuing analyses. There must be minimal non-specific binding of the DNA to the surface. In addition, the immobilization process should not interfere with the ability of the immobilized probes to hybridize and to be substrates for enzymatic solid phase synthesis. For the majority of applications, it is best for only one point of the DNA to be immobilized, ideally a terminus.
In recent years, a number of methods for the covalent immobilization of DNA to solid supports have been developed which attempt to meet all the criteria listed above. For example, appropriately modified DNA has been covalently attached to flat surfaces functionalized with amino acids (Running, J. A., and Urdea, M. S. (1990) Biotechniques, 8, 276-277), (Newton, C. R., et al., (1993) Nucl. Acids. Res., 21, 1155-1162.), (Nikiforov, T. T., and Rogers, Y. H. (1995) Anal. Biochem., 227, 201-209), carboxyl groups, (Zhang, Y., et al., (1991) Nucl. Acids. Res., 19 3929-3933), epoxy groups (Lamture, J. B. et al., (1994) Nucl. Acids. Res., 22, 2121-2125), (Eggers, M. D., et al., (1994) BioTechniques, 17, 516-524) or amino groups (Rasmussen, S. R., et al., (1991) Anal. Biochem., 198, 138-142). Although many of these methods were quite successful for their respective applications, the density of oligonucleotide bound (maximum of approximately 20 fmol of DNA per square millimeter of surface) (Lamture, J. B., et al., (1994) Nucl. Acids. Res. 22, 2121-2125), (Eggers, M. D., et al., (1994) BioTechniques, 17, 516-524), was far less than the theoretical packing limit of DNA.
Therefore, a method for achieving higher densities of immobilized nucleic acids on a surface is needed. In particular, a method for achieving higher densities of surface immobilized nucleic acids which permits use, manipulation and further reaction of the immobilized nucleic acids, as well as analysis of the reactions, is needed.
In connection with the need for improved nucleic acid immobilization methods for use, for example, in analytical and diagnostic systems, is the need to develop sophisticated laboratory tools that will automate and expedite the testing and analysis of biological samples. At the forefront of recent efforts to develop better analytical tools is the goal of expediting the analysis of complex biochemical structures. This is particularly true for human genomic DNA, which is comprised of at least about one hundred thousand genes located on twenty four chromosomes. Each gene codes for a specific protein, which fulfills a specific biochemical function within a living cell. Changes in a DNA sequence are known as mutations and can result in proteins with altered or in some cases even lost biochemical activities; this in turn can cause a genetic disease. More than 3,000 genetic diseases are currently known. In addition, growing evidence indicates that certain DNA sequences may predispose an individual to any of a number of genetic diseases, such as diabetes, arteriosclerosis, obesity, certain autoimmune diseases and cancer. Accordingly, the analysis of DNA is a difficult but worthy pursuit that promises to yield information fundamental to the treatment of many life threatening diseases.
Unfortunately, the analysis of DNA is made particularly cumbersome due to size and the fact that genomic DNA includes both coding and non-coding sequences (e.g., exons and introns). As such, traditional techniques for analyzing chemical structures, such as the manual pipeting of source material to create samples for analysis, are of minimal value. To address the scale of the necessary analysis, scientists have developed parallel processing protocols for DNA diagnostics.
For example, scientists have developed robotic devices that eliminate the need for manual pipeting and spotting by providing a robotic arm that carries at its proximal end a pin tool device that consists of a matrix of pin elements. The individual pins of the matrix are spaced apart from each other to allow each pin to be dipped within a well of a microtiter plate. The robotic arm dips the pins into the wells of the microtiter plate thereby wetting each of the pin elements with sample material. The robotic arm then moves the pin tool device to a position above a target surface and lowers the pin tool to the surface contacting the pins against the target to form a matrix of spots thereon. Accordingly, the pin tool expedites the production of samples by dispensing sample material in parallel.
Although this pin tool technique works well to expedite the production of sample arrays, it suffers from several drawbacks. First during the spotting operation, the pin tool actually contacts the surface of the substrate. Given that each pin tool requires a fine point in order that a small spot size is printed onto the target, the continuous contact of the pin tool against the target surface will wear and deform the fine and delicate points of the pin tool. This leads to errors which reduce accuracy and productivity.
An alternative technique developed by scientists employs chemical attachment of sample material to the substrate surface. In one particular process, DNA is synthesized in situ on a substrate surface to produce a set of spatially distinct and diverse chemical products. Such techniques are essentially photolithographic in that they combine solid phase chemistry, photolabile protecting groups and photo activated lithography. Although these systems work well to generate arrays of sample material, they are chemically intensive, time consuming, and expensive.
It is further troubling that neither of the above techniques provide sufficient control over the volume of sample material that is dispensed onto the surface of the substrate. Consequently, error can arise from the failure of these techniques to provide sample arrays with well controlled and accurately reproduced sample volumes. In an attempt to circumvent this problem, the preparation process will often dispense generous amounts of reagent materials. Although this can ensure sufficient sample volumes, it is wasteful of sample materials, which are often expensive and of limited availability.
Even after the samples are prepared, scientists still must confront the need for. sophisticated diagnostic methods to analyze the prepared samples. To this end, scientists employ several techniques for identifying materials such as DNA. For example, nucleic acid sequences can be identified by hybridization with a probe which is complementary to the sequence to be identified. Typically, the nucleic acid fragment is labeled with a sensitive reporter function that can be radioactive, fluorescent, or chemiluminescent. Although these techniques can work well, they do suffer from certain drawbacks. Radioactive labels can be hazardous and the signals they produce decay over time. Nonisotopic (e.g. fluorescent) labels suffer from a lack of sensitivity and fading of the signal when high intensity lasers are employed during the identification process. In addition, labeling is a laborious and time consuming error prone procedure. Consequently, the process of preparing and analyzing arrays of a biochemical sample material is complex and error prone.
Therefore, it is an object herein to provide improved systems and methods for preparing arrays of sample material. It is a further object to provide systems that allow for the rapid production of sample arrays. It is a further object herein to provide supports to which high densities of nucleic acids molecules are linked.
Processes for immobilizing a high density of nucleic acids on a surface, which are based on rapidly reacting a free thiol group of a modified surface or modified nucleic acid, under appropriate conditions, with a thiol-reactive functionality of the other component (surface or nucleic acid) are provided. This reaction may be direct or through a bifunctional cross-linking reagent. In a preferred embodiment, the modified nucleic acid includes a thiol group and the cross-linking reagent contains an iodoacetyl group.
Solid supports to which are linked xe2x80x9cbeadsxe2x80x9d which are linked to nucleic acid molecules are also provided. The beads are not necessarily spherical, but refer to particles that are conjugated to the solid support to thereby increase the surface area of the solid support and/or to provide an alternative surface for conjugation of nucleic acids or other molecules. The beads are preferably of a size of about 1 xcexcm to 100 xcexcm. Compositions containing at least one bead conjugated to a solid support and further conjugated to at least one molecule, particularly a nucleic acid are provided. The bead is formed from any suitable matrix material known to those of skill in the art, including those that are swellable and nonswellable. The solid support is any support known to those of skill in the art for use as a support matrix in chemical syntheses and analyses. In such instances, the nucleic acid is linked to the xe2x80x9cbeadxe2x80x9d via a sulfur atom as described herein. In certain embodiments, the beads may be conjugated on the solid support in wells or pits on the surface, or the beads may be arranged in the form of an array on the support.
Preferably the bead is made of a material selected from materials that serve as solid supports for synthesis and for assays including but not limited to: silica gel, glass, magnet, polystyrene/1% divinylbenzene resins, such as Wang resins, which are Fmoc-amino acid-4-(hydroxy-methyl)phenoxymethylcopoly(styrene-1% divinylbenzene (DVD)) resin, chlorotrityl (2-chlorotritylchloride copolystyrene-DVB resin) resin, Merrifield (chloromethylated copolystyrene-DVB) resin metal, plastic, cellulose, cross-linked dextrans, such as those sold under the tradename Sephadex (Pharmacia) and agarose gel, such as gels sold under the tradename Sepharose (Pharmacia), which is a hydrogen bonded polysaccharide-type agarose gel, and other such resins and solid phase supports known to those of skill in the art. In a preferred embodiment, the bead is of a size in the range of about 0.1 to 500 xcexcm, more preferably about 1 to 100 xcexcm, in diameter.
The solid support is in any desired form, including, but not limited to: a bead, capillary, plate, membrane, wafer, comb, pin, a wafer with pits, an array of pits or nanoliter wells and other geometries and forms known to those of skill in the art.
In another aspect, kits for immobilized nucleic acids on an insoluble support are provided. In one embodiment, the kit can comprise an appropriate amount of: i) a thiol-reactive cross-linking reagent; and ii) a surface-modifying reagent for modifying a surface with functionality which can react with the thiol-reactive cross-linking reagent. The kit can optionally include an insoluble support, e.g., a solid surface, magnetic microbeads or silicon wafers, for use in immobilizing nucleic acids. The kit can also optionally include appropriate buffers as well as instructions for use.
Use of these processes for immobilizing nucleic acid molecules onto a solid support results in at least 12.5-fold higher immobilization than previously reported techniques. The processes are therefore particularly useful for forming nucleic acid launching pads for mass spectrometry.
The nucleic acids immobilized on a surface using the methods provided herein can be used in a variety of solid phase nucleic acid chemistry applications, including but not limited to nucleic acid synthesis (chemical and enzymatic), hybridization and/or extension, and in diagnostic methods based in nucleic acid detection and polymorphism analyses (see, e.g., U.S. Pat. No. 5,605,798). Accordingly, further provided herein are methods of reacting nucleic acid molecules in which the nucleic acid molecules are immobilized on a surface either by reacting a thiol-containing derivative of the nucleic acid molecule with an insoluble support containing a thiol-reactive group or by reacting a thiol-containing insoluble support with a thiol-reactive group-containing derivative of the nucleic acid molecule and thereafter further reacting the immobilized nucleic acid molecules.
In a particular embodiment of the methods of reacting immobilized nucleic acids, the immobilized nucleic acid is further reacted by hybridizingxwith a nucleic acid that is complementary to the immobilized nucleic acid or a portion thereof. Such hybridization reactions can be used to detect the presence of a specific nucleic acid in a sample. This is of particular use in the detection of pathogens in a sample, such as a biological sample, that may be employed in the diagnosis of diseases.
Therefore, also provided herein are methods of detecting a target nucleic acid in a sample wherein a thiol-containing nucleic acid complementary to the target nucleic acid is immobilized to a surface using the processes described herein and the sample is contacted with the surface under conditions whereby target nucleic acid in the sample hybridizes to the immobilized nucleic acid. The hybridized target nucleic acid may be detected using a variety of methods, the preferred method being mass spectrometry. Further provided herein are methods of detecting alterations (e.g., deletions, insertions and conversions) in the nucleotide sequence of the target nucleic acid. In these methods, the molecular weight of the hybridized target nucleic acid, as determined by mass spectrometry, is compared to the molecular weight expected for the target nucleic acid sequence. Deviations of the measured molecular weight from the expected molecular weight are indicative of an alteration in the nucleotide sequence of the target nucleic acid.
In other methods of detecting a target nucleic acid in a sample as provided herein, the target nucleic acid is immobilized to a surface containing thiol-reactive groups. In these methods, prior to immobilization, the target nucleic acid is amplified in a reaction in which an oligonucleotide primer contains a 3xe2x80x2- or 5xe2x80x2-disulfide linkage and the resulting product is reduced to generate a thiol-containing nucleic acid. The thiol-containing nucleic acid is immobilized to a surface containing thiol-reactive groups and is contacted with a single-stranded nucleic acid that is complementary to the immobilized nucleic acid or a portion thereof. Hybridization of the single-stranded nucleic acid may be detected by a variety of methods. For example, the single-stranded nucleic acid may be labeled with a readily detectable moiety, e.g., radioactive or chemiluminescent labels. In a preferred embodiment, the single-stranded nucleic acid is detected by mass spectrometry.
In another embodiment of the methods of reacting immobilized nucleic acids, the immobilized nucleic acid is further reacted by extension of a nucleic acid that is hybridized to the immobilized nucleic acid or a portion thereof. Extension reactions such as these can be used, for example, in methods of sequencing DNA molecules that are immobilized to an insoluble support using the processes described herein. Thus, also provided herein are methods of determining the sequence of a DNA molecule on a substrate in which a thiol-containing derivative of the DNA molecule is immobilized on the surface of an insoluble support containing thiol-reactive groups and hybridized with a single-stranded nucleic acid complementary to a portion of the immobilized DNA molecule prior to carrying out DNA synthesis in the presence of one or more dideoxynucleotides.
Extension of a nucleic acid primer that is hybridized to a nucleic acid immobilized to a surface as provided herein also can be used in the detection of nucleotide sequence alterations (e.g., deletions, insertions, conversions) of a target nucleic acid. Accordingly, provided herein are methods of detecting alterations in a target nucleic acid sequence in which a single-stranded nucleic acid is hybridized to a thiol-containing target nucleic acid immobilized to a solid support according to the processes provided herein and the hybridized single-stranded nucleic acid is extended by addition of nucleotides to the 3xe2x80x2 end of the molecule. The extension product is characterized by, for example, mass spectrometry to determine whether its characteristics differ from those expected of a sequence complementary to the immobilized target nucleic acid. Thus, for example, the molecular weight of the extension product determined by mass spectrometry is compared to the expected molecular weight of a nucleic acid complementary to the target nucleic acid. Deviations from the expected molecular weight are indicative of an alteration in the sequence of the target nucleic acid.
In particular embodiments of the methods of detecting alterations in a target nucleic acid sequence provided herein, the target nucleic acid may be amplified prior to immobilization to a thiol-reactive surface in a reaction in which an oligonucleotide primer contains a 3xe2x80x2- or 5xe2x80x2-disulfide linkage. The resulting product is reduced to generate a thiol-containing target nucleic acid. The thiol-containing target nucleic acid is then immobilized to a surface containing thiol-reactive groups and the single-stranded complementary nucleic acid is hybridized thereto and extended.
In a further embodiment of the methods of detecting alterations in a target nucleic acid sequence provided herein, a single-stranded nucleic acid complementary to the target nucleic acid is immobilized to a surface through a linkage that includes a thiol group-thiol reactive functionality bond and a cleavable linker moiety. The sample containing target nucleic acid is contacted with the surface under conditions whereby the target hybridizes with the immobilized single-stranded nucleic acid. The immobilized single-stranded nucleic acid is extended by addition of nucleotides to the 3xe2x80x2 end of the molecule. Following extension, the double-stranded molecule is denatured and the single-stranded immobilized extension product is cleaved from the surface at the position of the linker. The extension product is characterized by, for example, mass spectrometry to determine whether its characteristics differ from those expected of a sequence complementary to the immobilized target nucleic acid.
It is understood that all applications of the solid phase nucleic acid chemistry based on nucleic acids immobilized to a solid substrate according to the processes provided herein can be conducted with thiol-containing nucleic acids and a thiol-reactive surface as well as with thiol-reactive nucleic acids and a thiol-containing support.
Methods of forming an array of nucleic acids on a surface of a substrate by contacting thiol-containing nucleic acids with an insoluble support containing thiol-reactive groups positioned in an ordered arrangement on the surface of the support are also provided herein. In an alternative method of forming an array of nucleic acids on a surface of a substrate as provided herein, an insoluble support containing thiol functionalities positioned in an ordered arrangement on the surface of the support is contacted with nucleic acids containing a thiol-reactive group.
Further provided herein are systems and methods for preparing a sample for analysis, and more specifically to systems and methods for dispensing low volumes of fluid material onto a substrate surface for generating an array of samples for diagnostic analysis. Systems and methods provided herein for preparing arrays of sample material are generally less expensive to employ and conserve reagent materials while allowing for the rapid production of highly reproducible sample arrays.
Provided herein with respect to systems and methods for dispensing low volumes of fluid material onto a substrate surface are serial and parallel dispensing tools that can be employed to generate multi-element arrays of sample material on a substrate surface. The substrate surfaces can be flat or geometrically altered to include wells of receiving material.
In one embodiment, the tool is one that allows the parallel development of a sample array. To this end, the tool can be understood as an assembly of vesicle elements, or pins, wherein each of the pins can include a narrow interior chamber suitable for holding nanoliter volumes of fluid. Each of the pins can fit inside a housing that itself has an interior chamber. The interior housing can be connected to a pressure source that will control the pressure within the interior housing chamber to regulate the flow of fluid through the interior chamber of the pins. This allows for the controlled dispensing of defined volumes of fluid from the vesicles.
In an alternative embodiment, the tool includes a jet assembly that can include a capillary pin having an interior chamber, and a transducer element mounted to the pin and capable of driving fluid through the interior chamber of the pin to eject fluid from the pin. In this way, the tool can dispense a spot of fluid to a substrate surface by spraying the fluid from the pin. Alternatively, the transducer can cause a drop of fluid to extend from the capillary so that fluid can be passed to the substrate by contacting the drop to the surface of the substrate.
Further, the tool can form an array of sample material by dispensing sample material in a series of steps, while moving the pin to different locations above the substrate surface to form the sample array. In a further embodiment, the prepared sample arrays are passed to a plate assembly that disposes the sample arrays for analysis by mass spectrometry. To this end, a mass spectrometer is provided that generates a set of spectra signal which can be understood as indicative of the composition of the sample material under analysis.
In one aspect, the dispensing apparatus provided herein for dispensing defined volumes of fluid, including nanovolumes and sub-nanovolumes of fluid, in chemical or biological procedures onto the surface of a substrate can include a housing having a plurality of sides and a bottom portion having formed therein a plurality of apertures, the walls and bottom portion of the housing defining an interior volume; one or more fluid transmitting vesicles, or pins, mounted within the apertures, having a nanovolume sized fluid holding chamber for holding nanovolumes of fluid, the fluid holding chamber being disposed in fluid communication with the interior volume of the housing, and a dispensing element that is in communication with the interior volume of the housing for selectively dispensing nanovolumes of fluid from the nanovolume sized fluid transmitting vesicles when the fluid is loaded into the fluid holding chambers of the vesicles. As described herein, this allows the dispensing element to dispense nanovolumes of the fluid onto the surface of the substrate when the apparatus is disposed over and in registration with the substrate.
In one embodiment the fluid transmitting vesicle has an open proximal end and a distal tip portion that extends beyond the housing bottom portion when mounted within the apertures. In this way the open proximal end can dispose the fluid holding chamber in fluid communication with the interior volume when mounted with the apertures. Optionally, the plurality of fluid transmitting vesicles are removably and replaceably mounted within the apertures of the housing, or alternatively can include a glue seal for fixedly mounting the vesicles within the housing.
In one embodiment the fluid holding chamber includes a narrow bore dimensionally adapted for being filled with the fluid through capillary action, and can be sized to fill substantially completely with the fluid through capillary action.
In one embodiment, the plurality of fluid transmitting vesicles comprise an array of fluid delivering needles, which can be formed of metal, glass, silica, polymeric material, or any other suitable material.
In one embodiment the housing can include a top portion, and mechanical biasing elements for mechanically biasing the plurality of fluid transmitting vesicles into sealing contact with the housing bottom portion. In one particular embodiment, each fluid transmitting vesicle has a proximal end portion that includes a flange, and further includes a seal element disposed between the flange and an inner surface of the housing bottom portion for forming a seal between the interior volume and an external environment. The biasing elements can be mechanical and can include a plurality of spring elements each of which is coupled at one end to the proximal end of each of the plurality of fluid transmitting vesicles, and at another end to an inner surface of the housing top portion. The springs can apply a mechanical biasing force to the vesicle proximal end to form the seal.
In a further embodiment, the housing further includes a top portion, and securing element for securing the housing top portion to the housing bottom portion. The securing element can comprise a plurality of fastener-receiving apertures formed within one of the top and bottom portions of the housing, and a plurality of fasteners for mounting within the apertures for securing together the housing top and bottom portions.
In one embodiment the dispensing element can comprise a pressure source fluidly coupled to the interior volume of the housing for disposing the interior volume at a selected pressure condition. Moreover, in an embodiment wherein the fluid transmitting vesicles are filled through capillary action, the dispensing element can include a pressure controller that can vary the pressure source to dispose the interior volume of the housing at varying pressure conditions. This allows the controller varying element to dispose the interior volume at a selected pressure condition sufficient to offset the capillary action to fill the fluid holding chamber of each vesicle to a predetermined height corresponding to a predetermined fluid amount. Additionally, the controller can further include a fluid selection element for selectively discharging a selected nanovolume fluid amount from the chamber of each vesicle. In one particular embodiment, a pressure controller is included that operates under the controller of a computer program operating on a data processing system to provide variable control over the pressure applied to the interior chamber of the housing.
In one embodiment the fluid transmitting vesicle can have a proximal end that opens onto the interior volume of the housing, and the fluid holding chamber of the vesicles are sized to substantially completely fill with the fluid through capillary action without forming a meniscus at the proximal open end. Optionally, the apparatus can have plural vesicles, wherein a first portion of the plural vesicles include fluid holding chambers of a first size and a second portion including fluid holding chambers of a second size, whereby plural fluid volumes can be dispensed.
In another embodiment, the dispensing apparatus can include a fluid selection element that has a pressure source coupled to the housing and in communication with the interior volume for disposing the interior volume at a selected pressure condition, and an adjustment element that couples to the pressure source for varying the pressure within the interior volume of the housing to apply a positive pressure in the fluid chamber of each of the fluid transmitting vesicles to vary the amount of fluid dispensed therefrom. The selection element and adjustment element can be computer programs operating on a data processing system that directs the operation of a pressure controller connected to the interior chamber.
In a further alternative embodiment, the apparatus provided herein is for dispensing a fluid in chemical or biological procedures into one or more wells of a multi-well substrate. The apparatus can include a housing having a plurality of sides and a bottom portion having formed therein a plurality of apertures, the walls and bottom portion defining an interior volume, a plurality of fluid transmitting vesicles, mounted within the apertures, having a fluid holding chamber disposed in communication with the interior volume of the housing, and a fluid selection and dispensing means in communication with the interior volume of the housing for variably selecting am amount of the fluid loaded within the fluid holding chambers of the vesicles to be dispensed from a single set of the plurality of fluid transmitting vesicles. Accordingly, the dispensing means dispenses a selected amount of the fluid into the wells of the multi-well substrate when the apparatus is disposed over and in registration with the substrate.
In yet another embodiment, provided herein is a fluid dispensing apparatus for dispensing fluid in chemical or biological procedures into one or more wells of a multi-well substrate, that comprises a housing having a plurality of sides and top and bottom portions, the bottom portion having formed therein a plurality of apertures, the walls and top and bottom portions of the housing defining an interior volume, a plurality of fluid transmitting vesicles, mounted within the apertures, having a fluid holding chamber sized to hold nanovolumes of the fluid, the fluid holding chamber being disposed in fluid communication with the volume of the housing, and mechanical biasing element for mechanically biasing the plurality of fluid transmitting vesicles into sealing contact with the housing bottom portion.
General methods for preparing an array of sample material on a surface of a substrate as described herein include the steps of providing a vesicle having an interior chamber containing a fluid, disposing the vesicle adjacent a first location on the surface of the substrate, controlling the vessel for delivering a nanoliter volume of a fluid at the first location of the surface of the substrate, and moving the vesicle to a set of positions adjacent to the surface substrate whereby fluid is dispensed at each location of the set of positions for forming an array of sample material;
Substrates employed during the general processes of preparing an array of sample material described herein can include flat surfaces for receiving the sample material as well as having the surfaces that include wells formed on the surface for defining locations for receiving the fluid that can be ejected from the chambers of the vesicles. Such substrates can be silicon, metal, plastic, a membrane, polymeric material, a metal-grafted polymer, as well as a substrate that is functionalized chemically, functionalized with beads, functionalized with dendrite trees of captured material, or any combinations of the above or any similar suitable material for receiving the dispensed fluid.
It is understood that in the general methods for preparing an array of sample material on a substrate surface described herein the apparatus can dispense both an analyte material as well as a support material, such as a matrix material, that aids in the analysis of the analyte. To this end the methods provided herein can include the steps of depositing a matrix material onto the substance of the substrate. Further the methods can also include a step of waiting a predetermined period of time to allow a solvent of the matrix material to evaporate. Once the solvent of the matrix material has evaporated, the methods herein can include a step of ejecting a volume of analyte fluid into the evaporated matrix material to dissolve with the matrix material and to form a crystalline structure on the substrate surface. It is understood that this step of redissolving the matrix material with the analyte material aids in the analysis of the composition of the material during certain analytical processes, such as mass spectrometry.
In an alternative practice, the methods herein can include a step of dispensing a mixture that consists of the analyte material and the matrix material, as well as other material compositions. In this way the matrix and the analyte are delivered to the surface of the substrate as one volume of material. In a further step, the prepared arrays of sample material can be provided to a diagnostic tool for determining information that is representative of the composition of the sample material.
Once such diagnostic tool can include a mass spectrometer. The mass spectrometers can be time of flight mass spectrometers, Fourier transform mass spectrometers or any other suitable type of mass spectrometer that allows the analysis of composition of the sample array.
In one practice of the methods, the step of providing a vesicle having an interior chamber includes the step of providing a vesicle having a piezo electric element for causing fluid to move through the chamber. This method can also include the step of moving the vesicle by rasterizing the vesicle across the surface of the substrate, to form the array of sample material.
In an alternative practice of the methods, parallel processing protocols can be employed wherein the vesicle that is employed during the processing includes a vesicle assembly that has a plurality of vesicles arranged into a matrix for dispensing fluid to a first plurality of locations on the substrate surface. In this way in a single operation, the method provides for forming a matrix of a sample material on the substrate surface. Offset printing can also be employed to form a large matrix of sample material by employing multiple printing steps with the vesicle matrix. Other printing techniques can be employed by the present invention without departing from the scope thereof.
In another embodiment, fluid can be dispensed to the surface of the substrate by contacting the vesicle against the surface of the substrate to spot the surface of the substrate with sample material. Alternatively, the methods provide for another non-contact printing approach wherein the processes of the invention cause a drop of fluid to be formed on at the distal tip of the vesicle. It is the drop of fluid that is contacted against the surface of the substrate for delivering sampling material thereto. This provides for the controlled delivery for the known volume of fluid without resulting in the contacting of the vesicle against the surface of the substrate.
In further embodiments, vesicles are provided having an interior chamber that is dimensionally adapted to allow filling of the chamber by capillary action.
In another aspect, methods are provided for analyzing a material, that comprise the steps of providing a vesicle suitable for carrying a fluid having the material therein, disposing the vesicle adjacent a first location of the surface of the substrate, controlling the vesicle to deliver a nanoliter volume of the fluid to provide a defined and controlled volume of fluid at the first location of the surface of the substrate, moving the vesicle to a second position adjacent a second location on the surface on the substrate to dispense a defined and controlled volume of the material along an array of locations along the substrate surface, and performing mass spectrometry analysis of the material at each location of the array. These methods can include the step of mixing a matrix material and an analyte material to form the fluid being delivered to the substrate surface. Alternatively, this embodiment can include the steps of filling a chamber contained within the vesicle with a matrix material and dispensing the matrix material to the array of locations. Subsequently, analyte can be dispensed. The step of performing mass spectrometry can include the step of performing a matrix assisted laser desorption ionization mass spectrometry, as well as time of flight mass spectrometry, or a Fourier transform spectrometry.
In another aspect, apparatus for forming an array of a sample material on a surface of a substrate are provided. Such apparatus will compromise a vesicle having a distal end suitable for carrying a fluid thereon, a movable arm having a distal portion mounted to the vesicle, a controller for moving the arm to dispose the vesicle adjacent a first location on the surface on the substrate and for controlling the vesicle to provide a nanoliter volume of the fluid at the first location of the surface of the substrate, and a diagnostic tool for analyzing the material to generate a composition signal that is representative of the chemical composition of the material. In this apparatus the vesicle can compromise a solid shaft of material as well as a vesicle having an interior chamber suitable for carrying fluid as well as a chamber for carrying a fluid in a transducer element for ejecting fluid from that chamber.
Further provided herein are substrates having a surface for carrying an array of a matrix material and formed according to a process comprising the steps of a providing a vesicle suitable for transferring a fluid containing a matrix material, disposing the vesicle adjacent a first location on the surface on the substrate, controlling the vesicle to deliver the fluid to the first location of the surface of the substrate, and moving a vesicle to a set of positions adjacent the surface of the substrate and delivering fluid at each of these locations to form an array of matrix material. This substrate itself can be a flat silicon chip as well as a any other suitable material, and can be pitted, include wells, and have wells that have rough interior surfaces.
In particular embodiments, the methods of forming an array of nucleic acids on a surface of a substrate as provided herein include contacting predetermined positions of the surface of an insoluble support with thiol-containing nucleic acid solutions dispensed to the positions with a vesicle having an interior chamber containing the respective solutions whereby the predetermined positions incorporate thiol-reactive groups. Alternatively, the entire surface of the substrate is derivatized with the thiol-reactive groups and thiol-containing nucleic acid is dispensed to predetermined positions on the surface in an array-forming manner. Also provided herein are substrates having a surface carrying an array of nucleic acids formed by the methods described herein.
The above and further features and advantages of the instant invention will become clearer from the following Figures, Detailed Description and Claims.